Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

A method for the construction of arrays from self-assembling monolayers
is described. The arrays have particular utility for the screening of
peptides ligands that can foster the growth of cells in culture. This
technique has been used to identify peptide ligands that foster the
growth of human stem cells, which otherwise require an extracellular
matrix in order to grow in an undifferentiated state. This also makes
possible an assay to identify other such peptides.

Claims:

1. A human stem cell culture comprising: human stem cells, a medium in
which human stem cells will grow, and a self-assembled monolayer support
presenting to the human stem cells, in a uniform orientation, molecules
of a peptide species that supports the growth of human stem cells in an
undifferentiated state.

5. A method for culturing undifferentiated human stem cells, the method
comprising the steps of: applying the stem cells and the medium to a
self-assembled monolayer support presenting to the human stem cells, in a
uniform orientation, molecules of a peptide species that supports the
growth of human stem cells in an undifferentiated state; and culturing
the cells under conditions that favor the growth of the cells.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Utility patent
application Ser. No. 11/504,573, filed Aug. 15, 2006 which claims the
benefit of U.S. Provisional Patent Application No. 60/708,167, filed Aug.
15, 2005, each of which is incorporated herein by reference as if set
forth in its entirety.

BACKGROUND

[0003] Stem cells are defined as cells that are capable of a
differentiation into many other differentiated cell types. Embryonic stem
cells are stem cells from embryos that are capable of differentiation
into most, if not all, of the differentiated cell types of a mature body.
Stem cells are referred to as pluripotent, which describes this
capability of differentiating into many cell types. A category of
pluripotent stem cell of high interest to the research community is the
human embryonic stem cell, abbreviated herein as hES cell, which is an
embryonic stem cell derived from a human embryonic source. hES cells are
of great scientific interest because they are capable of indefinite
proliferation in culture and are thus capable, at least in principle, of
supplying cells and tissues for replacement of failing or defective human
tissue. Methods to culture human embryonic stem cells offer the potential
of unlimited amounts of human cells and tissues for use in a variety of
therapeutic protocols to assist in human health. It is envisioned that in
the future hES cells will be proliferated and directed to differentiate
into specific lineages so as to develop differentiated cells or tissues
that can be transplanted into human bodies for therapeutic purposes.

[0004] One of most significant features of hES cells is the attribute of
being capable of self-renewal. By that it is meant that the hES cells are
capable of proliferating into multiple progeny stem cells, each of which
seems to have the full potential of its ancestor cell. In other words,
the progeny are renewed to have all the developmental and proliferative
capacity of the parental cell. This attribute, combined with the
pluripotency, are the traits that make hES cells candidates for many
potential uses, since, in theory, hES cells can be reproduced
indefinitely and in large numbers and then induced to become any cell
type in the human body. The attribute of ability to self-renew appears
closely linked to the attribute of being undifferentiated in the sense
that at least given present knowledge, only undifferentiated hES cells
are capable of indefinite self-renewal and as soon as the cells
differentiate, the attribute of self-renewal capability is lost. Since
hES cells will spontaneously differentiate, care must be taken in culture
conditions to maintain the cells in an undifferentiated state.

[0005] Among the factors that have so far been identified as successful in
maintaining hES cells in long term culture in an undifferentiated state
are the medium in which the cells are grown and the substrate on which
they are grown. Much progress has been made in defining media, which can
be formulated to include the activators of FGF and TGF-beta pathway and
suppressors of BMP and WNT pathways, which have the effect of enhancing
the cells self-renewal. Considerably less information is available on the
role of substrates and cell-substrate adhesion in hES cell survival and
growth.

[0006] In the original co-culture experiments, hES cells were plated on a
gelatin-coated surface containing mouse embryonic fibroblasts (MEFs) or
other feeder cells. Thomson et al., Science 282, 1145-1147 (1998). Upon
plating, it was found that the hES cells do not grow on top of feeder
cells, but instead tend to occupy the exposed gelatin-coated surface. As
the hES cells proliferate, these feeder cells are "pushed away" by the
growing ES cell colony. Imreh et al., Stem Cells and Development, 13,
337-343 (2004). This observation suggests that the gelatin-coated surface
along with the secreted factors provide a sufficient platform for the
attachment. It was also discovered that growth of cells on feeder layers
can be avoided through the use of "conditioned medium" or CM, which is
medium in which feeder cells have been cultured. However, culture of hES
cells on simple gelatin-coated surfaces even in CM leads to rapid
differentiation of the cells. Xu et al., Nat. Biotechnol. 19, 971-974
(2001). To date, growth of undifferentiated ES cells without exposure to
feeder cells has been achieved on surfaces coated with lysed MEFs in a
medium containing FGF and LIF, laminin in MEF-CM, fibronectin in the
media containing LIF, bFGF and TGF-beta and Matrigel®-coated surface
in various media conditions. (e.g., Xu, supra; Amit et al., Biol. Reprod.
70, 837-845 (2004); Hoffman & Carpenter, Nat. Biotechnol. 23, 699-708
(2005)). Matrigel® is a commercially produced extracellular matrix
material. Interestingly, there have been other reports mentioning the
failure of ES cell culture in the presence of MEF-CM on surfaces coated
with fibronectin, laminin and Matrigel®. A recent review summarizing
advances in hES cell culture techniques attributes this variability to
multiple factors including variability in media formulations, variability
in feeder types used for CM production, batch-to-batch variability of the
attachment substrates or even variability between hES cell lines
including origin, passage number, karyotypic stability and epigenetic
status. (Hoffman, supra).

[0007] It is important not to neglect the role of substrate attachment for
successful hES cell growth. Identifying defined hES cell growth
conditions requires the identification of defined growth media and a
defined hES cell attachment surface. Screening well-defined surfaces in
an array format allows rapid identification of specific molecules that
promote hES cell adhesion. Because of the relatively small amount of the
materials (e.g., cells and media) required to screen for cell adhesion to
a surface microarray, the screen can be easily repeated and performed in
parallel for multiple ES cell types and media formulations. Thus, this
strategy offers a low-cost and rapid means to find defined conditions and
therefore tame the variability present in hES cell culture literature.

[0008] To date, several successful examples of multicomponent microarrays
in cell-based screens have been reported. Lam and co-workers fabricated a
peptide-array prepared by contact spotting of peptides onto
glyoxylyl-functionalized glass slides. Falsey et al., Bioconjug. Chem.
12, 346-353 (2001). These arrays were used to identify peptides promoting
adhesion of a specific cancer cell line. Bhatia and co-workers presented
fabrication of a microarray presenting combinations of several proteins
fabricated via contact printing onto acrylamide-coated glass slides. This
array was used to screen for protein combinations that assist the
differentiation of mouse ES cell into hepatocytes. Langer and coworkers
fabricated an array of polymeric materials by spotting combinations of
monomers onto a glass slide followed by in situ polymerization.
Subsequently, they used a collection of cells obtained by trypsinization
of embryonic bodies derived from hES cells and identified several
polymers promoting cell adhesion and differentiation in the presence of
retinoic acid. A similar approach was recently reported to screen for
materials promoting adhesion of the mesenchymal stem cells. However, no
reports to date have described screening for growth of undifferentiated
hES cells. Identifying such conditions is challenging: The
undifferentiated state of hES cells is easily disrupted by small changes
in their growth environment, an attribute that has hindered the
development of a reliable and reproducible assay to assess a variety of
growth conditions.

BRIEF SUMMARY

[0009] The present invention involves three interrelated concepts. The
first concept is the use of alkane thiols (ATs) to construct
self-assembled monolayers (SAMs) in an array. The second concept is to
use peptide ligands attached to the ATs in selected areas of the array to
test for ligands to which human embryonic stem cells will adhere. The
third concept is to identify peptide ligands that will support the
culturing of human embryonic stem cells on such arrays.

[0010] The present invention is thus summarized in one aspect as a
monolayer array that is a SAM of AT molecules to which is attached
selectively moieties intended either to encourage or discourage the
contact of solutions and the adherence of cells to particular defined
locations in the array.

[0011] The present invention is also summarized in a second aspect as a
method to identify and define which peptides can be used to support the
culture of hES cells by making an array from a SAM in which defined areas
of the array present different peptides to cells placed on the array
surface in order to determine which peptides support the growth of hES
cells in culture.

[0012] The present invention is also summarized in a third aspect as
defined peptides to which hES cells will adhere in a SAM array so as to
culture and localize the hES cell colonies.

[0013] Other objects, features and advantages of the present invention
will become apparent from the following specification.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0014] FIG. 1 illustrates the structure of ATs species used in a cell
adhesion screen for hES cells, and the results of hES cell growth on
various peptides in the array.

[0017] FIG. 4 is illustrates the arrangement of the array elements and the
AT species used in the array.

[0018] FIG. 5 illustrates one of the synthesis schemes in the material and
methods presented below.

[0019] FIG. 6 illustrates another step in the synthesis scheme presented
in the methods and materials below.

[0020] FIGS. 7 and 8 illustrate some of the chemistry for another
alternative within the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0021] What is described here first is a method to use SAM chemistry to
construct arrays that can be used to present ligands to cells plated onto
the array. The arrays are preferably constructed of SAMs such as those
formed using ATs. An advantage of using ATs is that they form
reproducible SAMs and surfaces. This attribute means that the array
elements will vary only because of the peptide or other binding element
presented on the array and not because of other bulk properties of the
array, such as topology. This method of array formation can be used to
identify specific ligands or epitopes that act to promote hES cell
binding or self-renewal. To construct such an array, we desired to
utilize a method to pattern the SAM array so that ligands would only be
presented to the cells in defined areas of the arrays and that other
areas of the array (the background areas) would resist both solvents and
cell presence.

[0022] The SAMs permit distinct formulations for the background areas of
the array and the elements of the array. The background areas between the
array elements have a property referred to as "solvophobic," or resistant
to the spreading of solvents. The background should also be cytophobic to
resist cellular attachment. In the array elements, we desired to be able
to spot areas that would present specific ligands or epitopes on their
surface. The ligands would often be peptides, but can also be small
organic molecules.

[0023] To satisfy these criteria, a strategy of forming a SAM was adopted
using AT chemistry on gold in all the areas of the array. For, example, a
background was formed of perfluoro-AT that was both cytophobic and
solvophobic. First, the surface was coated with a perfluoro-AT monolayer,
either leaving holes in the monolayer for the elements of the array or
creating holes in the monolayer for the elements of the array in a
subsequent step. In the elements of the array, AT species carrying
various ligands could then be attached to the substrate in the holes to
complete the monolayer and to present ligands for cellular attachment in
each element. The strategy here is to coat a gold surface with
perfluoro-AT, leaving or creating "holes" in the array into which AT
species with specific ligands, also referred to here as epitopes,
attached are then spotted. Methods for synthesizing the AT species for
both regions on the array are found in more detail in the examples below.

[0024] Each AT species may be thought of as having three important regions
or moieties. One region is at the basal end, which is an attachment
moiety intended to attach the monolayer species to the substrate. In the
case of the AT species, the attachment moiety is the thiol groups, which
attaches to a gold substrate. Other attachment groups can attach to other
substrates. Another region is the intermediate region, which is the
spacer moiety, and in this case the alkane. Other simple organic groups
can be used for the spacer as long as the resulting species are capable
of self-assembly in a monolayer. Lastly, the active group at the end of
the monolayer species is the ligand, which can be a group intended to
repel cells (cytophobic) or to bind cells (cytophilic). The ligands
intended to bind cells, or to test binding to cells, can be, but are not
limited to peptides.

[0025] This system offers the ability to determine the peptide ligands
that will foster cell growth for cell types that require extracellular
signals for desired growth properties. In particular, this system can be
used to test peptide ligands to identify those which help to foster
self-renewal and, thus, the undifferentiated growth of hES cells. Arrays
can be fabricated that contain differing peptide elements in AT-peptide
species spotted in different array elements. After depositing hES cells
on the array, the elements that support the growth of hES cell colonies
will preferentially present those peptides to which ES cells
preferentially bind and grow. This same strategy can be used to identify
and find differentiated progeny lineages from mixed populations of ES
cells and differentiated progeny cells of similar or varied type.

[0026] Thus, in this specification, we also describe how we have used a
SAM array format to identify surfaces that promote the self-renewal of
hES cells. These cells typically grow in colonies of various size
randomly distributed on growth substrate. Unlike most conventional cells
that are grown in culture, ES cells undergo rapid differentiation and/or
death if they are cultured as a confluent monolayer. Therefore, one of
the key factors in the ES cell culture is control over ES cell density.
However, when any cells are cultured in an array format they proliferate
independently in each array element and "density of the culture" is
different for each array element. Therefore, the duration of the
experiment in array format must be carefully balanced to avoid
false-negative outcomes that can arise when array elements present cells
at high-density. Subtle variations in cell density cannot be eliminated
in multi-component arrays, because each array element presents a slightly
different environment for cell proliferation. Still, this parameter can
be minimized if the initial cell distribution is uniform throughout the
array elements. Control over cell density can be readily achieved with a
majority of conventional cell lines; however, it is not trivial to do so
with hES cells.

[0027] Conventional array fabrication methods that utilize contact
printing of small molecules onto functionalized surfaces yield random
covalent or non-covalent immobilization of molecules onto the surface.
This methodology will not afford reliable results in ES cell-based
screens, due to poor control over array element geometry, ligand
distribution, concentration and orientation on the surface of the array.
In contrast, we have found that SAMs of ATs on gold, as discussed above,
provide a well-defined system allowing state-of-the-art control over
presentation of surface-immobilized molecules. We describe here an
approach to the fabrication of the arrays of SAMs, and we demonstrate its
utility for screening for cell adhesion and for the culture of human ES
cells. We also show this technology to be compatible with ES cell growth
conditions. Here we have also sought to extend this approach further and
have demonstrated that a SAM-based microarray presenting a library of
molecules can be used as a platform for discovery of novel, well-defined
surfaces able to support proliferation of ES cells.

[0028] The starting point for design of the library of small molecules
promoting ES-cell adhesion emerged upon careful examination of the
components of the effective substrate Matrigel®. As mentioned, hES
cells can be cultured on a Matrigel® substrate, but this isolated
substrate contains extracellular matrix murine proteins in indeterminate
quantities. Laminin, its major component, is known to interact with
collagen and entactin (the other two major components) through globular
domains and B2 short arm, respectively. Thus, these mixtures form a
polymeric network with an organized display of particular protein
subunits. The superiority of a Matrigel®-coated surface to a surface
coated with pure laminin further suggests the necessity for the control
over surface presentation of the binding domains of the proteins.
Therefore, we sought to mimic both molecular and spatial composition of
Matrigel® by assembling selected regions of the laminin on the
surface with the highest degree of control over the presentation of the
peptides. Extensive work has been done to map the biological activity of
the laminin to specific chains, domains, and even short amino acid
sequences. We have selected a variety of laminin-derived peptides known
to exhibit strong biological activity (i.e. cell adhesion, neurite
extension, angiogenesis, etc.) to construct the initial library.

[0029] The methods described herein have enormous potential that arises
from interweaving synthetic chemistry and flexibility of SAMs to generate
arrays for screening for ES cell adhesion. The exceptional degree of
control over peptide ligand presentation and surface density provided by
SAMs is useful for screening for any cell surface interactions and
optimizing substrates for cells sensitive to their growth environment.
Interactions of cells with their environment depend on surface
concentration and presentation of the binding partners. Alteration of
these parameters often influences the outcome of the cellular response.
Embryo development presents the most fascinating example in which
gradients of concentrations of the same effector molecules controls the
differentiation pathways and morphological changes in the different
spatial locations of the embryo. Simple knowledge of the structure of
binding partners is not sufficient to explain these phenomena. We
therefore contend that the next-generation of cell-based screening must
involve technology allowing control over parameters beyond structure of
the binding peptide. The SAM-based arrays presented here highlight the
feasibility of such high-throughput multivariable screen on the single
array surface.

[0030] Well-defined arrays of SAMs presenting small molecules present an
important addition to currently reported techniques used for cell-based
screening. Interaction of cells with SAMs presenting cell-binding
peptides depends on the presence of a particular receptor in the cell and
can be often attributed to a defined receptor-ligand pair. This
reductionism in screening for cell-substrate interaction contrasts other
approaches that involve a multitude of inseparable variables. For
example, different cell types can bind to an array of extracellular
matrix proteins utilizing a different set of receptors. This degeneracy
in protein-cell interactions is hard to resolve using protein arrays, and
it has to be addressed using other techniques. On the contrary, if they
exist, specific cell surface receptor partners for bulk polymeric
materials have yet to be identified; materials that incorporate
particular recognition epitopes can be used to guide cells reproducibly
to manifest specific states or behaviors. Although it is possible to
screen for a polymeric material exhibiting a particular biological
function, it is often impossible elucidate the mechanism for a particular
material-specific biological activity.

[0031] The screen presented in this report utilized known fragments of
laminin. Thus, the ability of some peptide sequences (e.g. RGD, YIGSR
(SEQ ID NO:1), GNRWHSIYITRFG (SEQ ID NO:7)) to support ES cell growth can
be explained using information about gene expression of their binding
partners (αvβ3 and αvβ5
integrins, 37/64 kDa receptor, α6β1 integrin) using
reported ES cell gene expression profiles. For small molecules with no
known binding partners, binding partners can be identified using
established biochemical techniques. This technique enables the
integration o f receptor identification techniques with screening
involving a large collection of small molecule ligands for specific
cell-surface receptors. This will allow identification of the ES cell
surface receptors involved in robust long-term growth and will suggest a
mechanism by which signals induced by substrate adhesion control
self-renewal of ES cells. Additionally, we have demonstrated that the
findings of our screen can be extended to the design of materials
supporting ES cell growth. Because the identified materials are fully
synthetic they present no risk of contamination with animal products, and
they can avoid problems of batch-to-batch variability of the materials of
biological origin. Therefore, this technology provides an important
contribution in the current quest for the state-of-the-art and
well-defined ES cell growth conditions.

EXAMPLES

[0032] General Preparation of the Arrays of Self-Assembled Monolayers
(Assembly After Conjugation Strategy): Chromium (1 nm) and then gold (25
nm) were evaporated onto piranha-cleaned glass coverslips (Corning No 1%,
23 mm squares) using a thermal evaporator (Denton Vacuum, Moorestown,
N.J.). Substrates were immediately immersed into a 1 mM solution of
fluoro-AT in absolute ethanol. After twenty-four hours, substrates were
thoroughly rinsed with ethanol and dried under a stream of nitrogen.
Coverslips with fluoro-AT SAM were irradiated with UV-light (1 kW, Hg--Xe
Research Arc Lamp, Oriel Instruments (Spectra-Physics, Stratford, Conn.)
through a quartz photomask (array of 500 μm or 750 μm squares,
0.067 quartz-chromium mask (Photo Sciences, Torrance, Calif.)) for 1
hour. Irradiated samples were rinsed thoroughly using several repetitive
washes with absolute ethanol and distilled water and dried under a steam
of nitrogen. Spotting of AT solutions onto the bare gold areas was
performed within two hours of the photolithography. Spotting was
performed manually using a P2-Pipetman (Gilson) in a humidity chamber.
Spotted arrays were stored in the humidity chamber for twelve hours and
thoroughly washed using repeated washes with ethanol and water. Rapid
flow during washing was used to prevent cross-contamination of array
spots.

[0033] Cell Culture: A neuroblastoma cell line, SH-SY5Y, was grown in 10%
heat-inactivated fetal bovine serum (HI FBS), 45% high glucose Dulbecco's
modified Eagle medium (DMEM), 45% Ham's F12 nutritional supplement (F12)
with penicillin and streptomycin in an atmosphere of 5% CO2 at
37° C. A fibroblast cell line, Swiss 3T3, was propagated in 10% HI
FBS in DMEM. For plating of the cells on the chips, cells were incubated
at 37° C. with 5% trypsin/ethylenediamine-tetraacetic acid (EDTA),
centrifuged and resuspended in media at 2×105 cells/mL and
5×105 cells/mL for the fibroblasts and neuroblastomas,
respectively. Chips were sterilized by placing each in a single well of a
6-well plate, soaking in 70% ethanol, and drying under UV light for 1
hour. To the individual chips was added 3 mL of the cell suspensions to
each well. After 2-5 hours, the media was gently replaced three times and
the chips were picked up, transferred to a clean well and 3 mL of media
was added. The cells on the chips were typically propagated between 1-7
days. The media was then removed, and the cells were fixed in PFA buffer
(2% paraformaldehyde, 350 mM NaCl, 150 mM HEPES, 10 mM CaCl2, pH
7.4) for 20 minutes at 0° C. The cells were washed twice with
Dulbecco's phosphate buffered saline and stained (0.1% Coomassie
Brilliant Blue in H2O/MeOH/AcOH (50:50:1)) for 5-15 minutes after
which the cells were washed twice with water and air dried. Images were
generated on a Leica MZ6 dissecting microscope.

[0035] Chips were sterilized as described above. To aid in ES cell
patterning, the chips were then soaked in DMEM/F12 with 15% defined FBS
for 1-2 hours and washed thoroughly. Then cells were plated on each chip
(1-2×106 cells/chip) and allowed to incubate overnight. The
next morning the chips were moved to fresh wells and fed with CM.

[0036] Test for alkaline phosphatase activity: The procedure for staining
for alkaline phosphatase activity was essentially identical to the
procedure supplied by the manufacturer. After propagation of the ES
cells, the media was removed. The reagents in 100 mM Tris pH 8.5 were
incubated with the chips for 30 minutes at 25° C. The chips were
washed gently for 5 min with Tris buffer and then fixed and dried as
usual.

[0037] Initial experiments to test fluoro-AT SAM cytophobicity and cell
patterning: Chips with a fluoro-AT SAM background and PEG acid-AT squares
fabricated by immersion (vide supra) were plated with both SH-SY5Y
neuroblastoma and Swiss-3T3 fibroblast cells that were either imaged as
live cells or as fixed and stained cells. For both cell types, dense
squares of cells on the cytophilic surface (PEG acid-AT) were formed. A
minimal number of cells growing on the background were observed, thereby
demonstrating the cytophobic property of the fluoro-AT SAM background.

[0038] Initial experiments to test cell response to SAM formation by
spotting: A chip with a fluoro-AT SAM background surrounding bare gold
squares was formed by photolithography. To this was spotted an aqueous
solution (1 mM) of cytophilic PEG acid-AT in a pattern of 3×3
squares. An ethanolic solution of fluoro-AT was spotted on the remaining
bare gold squares. Neuroblastoma SH-SY5Y cells were plated, proliferated,
fixed and stained. A pattern corresponding to cells affixed to only the
expected cytophilic surface was evident as was minimal binding to the
cytophobic surface. This result demonstrates that spotting solutions of
ATs can generate both cytophilic and cytophobic SAMs.

[0039] Solvophobicity Tests: Advancing contact angles were obtained for
each of the surfaces with various solvents. Briefly, a small volume of
solvent was added to the cleaned surfaces via syringe. As additional
solvent was added the angle was measured through a magnifying bezel.
Values from 180°-90° reflect a high degree of beading. As a
benchmark, values for mixtures of water with polar-organic solvents were
obtained for an undecanethiol SAM. (FIG. S2) A reduction in beading with
increasing amount of organic solvent was observed. Polar organic solvents
were chosen because these solvents dissolve most peptides and
biologically active small molecule::. Measured values for 100% DMSO and
50% DMF are 61° and 72° respectively. The fluoro-AT SAM was
the most effective at beading these solvents; this surface gives values
of 80° and 92° for 100% DMF and 100% DMSO respectively.

[0040] To test the feasibility of spotting arrays with a hand held
pipette, approximately 200 nL of water was spotted on the chips patterned
with 750 μm hydrophilic PEG acid-AT squares surrounded by fluoro-AT
SAM background. The water clearly formed beads due to the high contact
angle between the solvent and the fluorinated surface. The importance of
high contact angles of the background was reflected in that increased
beading allows a high density of spots on a single chip. The higher the
contact angle, more spots can be fit on the chip. A simple geometric
rationale shows that the contact radium (ρ) of the liquid drop with
volume (V) depended on the surface contact angle (θ) as:

ρ=sin-1θ [(π/3V)(1-cos θ)(2+cos
θ)]-1/3

[0041] No gravity or surface tension effects are included in this
analysis.

[0042] It was evident that the effective size (physical size) of a spot no
longer changes for contact angles above 90°. The relevance of the
solvophobicity of the fluoro-AT SAM background was that it provides
contact angles above or near to 90° for a wider range of solvents
than other hydrophobic surfaces and is also cytophobic.

[0043] To test the strategy for constructing arrays, we generated a
patterned array with an AT known to bind to cells generally, PEG acid-AT,
shown in FIG. 4, using cytophobic fluoro-AT as the background (also FIG.
4). Solutions of varying molar ratios of PEG acid-AT diluted with
polyol-AT, a hydrophilic AT that does not interact with cells, were
spotted on bare gold areas by photopatterning a SAM. Then, neuroblastoma
(SH-SY5Y) cells and fibroblast (Swiss 3T3) cells were plated on the
patterned surface, allowed to proliferate, fixed and stained. For both
cell lines, measured cell adhesion decreased as the mole fraction of the
acid in the spotting solutions approached 0.2. This indicates that the
spotting method worked and that SAMs composed of mixtures of AT species
can be screened as supports for cell cultures using this approach.

[0044] In physiological settings, cells interact with cues from the
extra-cellular matrix in which they reside. The sequence Arg-Gly-Asp
(RGD), found in a number of proteins, including fibronectin and
vitronectin, binds a family of integrins on the surface of some cell
lines, including the 3T3 fibroblasts. An Arg to Lys substitution in the
motif destroys the adhesion. Alternatively, the peptide YIGSR (SEQ ID
NO:1), a sequence found in the matrix protein laminin, interacts with the
neuroblastoma cell line but not with the fibroblasts. To test whether
surfaces that would preferentially react with defined cell lines could be
created, a surface displaying a fluoro-AT SAM was photopatterned.
Mixtures of polyol-AT and peptide-AT were then spotted on locations on
the array in the areas of bare gold. As controls, an AT species known to
bind cells generally, PEG acid-AT, was deposited in one element of the
array and an AT species known to resist cell binding generally,
polyol-AT, was deposited in another. Then neuroblastoma and fibroblast
cells were deposited on the array, allowed to proliferate, fixed and
stained. The fibroblasts bound to the RGD array element, bound minimally
to the KGD element and bound modestly to the YIGSR element. The
neuroblastoma cells adhered to the YIGSR element, but not to either of
the RGD or KGD elements. Thus, cell-specific adhesion is possible with
this system and specific to ligands for cell types.

[0045] To investigate the density issue, we ascertained how cell density,
cell survival and even array element size can influence cell distribution
in the array. A reverse time lapse analysis of ES cell aggregates
(clumps) grown on Matrigel® in CM for 48 hours. An overlay of the
colony outlines demonstrates that a typical colony originates from a very
limited number of cell aggregates. A majority of the plated hES cells
fail to attach and proliferate even on uniformly coated substrate. To
find the array element size yielding reproducible ES plating conditions,
we divided uniform substrate into areas of different size and estimated
the survival rate as a function of a size. Visually, the substrate
appears to be uniformly coated with cells one hour post plating. However,
an analysis of cell survival demonstrates that only 50% of colonies
deposited on the 0.2 mm square elements survived (or were viable) while
100% of the 0.8 mm elements contained viable colonies. The problem of
survival cannot be solved by a simple increase in cell density due to
problems associated with overgrowth-induced damage. Our analysis of
projected area of cell growth additionally demonstrates the effect of
cell aggregate size on survival rate. For cell clumps divided into large
(>40 cells), medium (10-40 cells) and small (5-10 cells), survival
rate in this particular example was 98%, 50% and 30%, respectively. This
variability in cell aggregate size and cell survival rate inevitably lead
to variability in the cell density in different array elements, even if
the array elements presented the cells with the same growth substrate.
For example, after 48 hours of proliferation cell, "density" ranges from
20% to 70% in different 0.8×0.8 mm elements.

[0046] This analysis underscores the importance of larger array element
size in ES screens. These data highlight how the current trend in the
technology towards miniaturization and fabrication of super high-density
arrays may not be relevant for some cell surface arrays used for ES
cells. With small array elements, ES cell survival will be stochastic,
even on array elements presenting ligands that support hES cell
self-renewal. When the size of the array elements is increased, those
elements that can support hES cell self-renewal can be more reproducibly
identified. Thus, screening results from arrays with elements that are
too small will be inconclusive. More importantly, this analysis
emphasizes the necessity for a high degree of control in array
fabrication for ES cell based screens. When dealing with cell culture
conditions for cells poised to undergo differentiation, it is important
to ensure that variability is a consequence of cell growth rather than
batch-to-batch reproducibility of the array.

[0047] We tested arrays of SAMs presenting a number of short peptides for
their ability to support the growth of undifferentiated hES cells. The
screen was done with the array presenting square elements of
0.5×0.5 mm (FIG. 1b) or 0.8×0.8 mm size (FIG. 1c). The
0.5×0.5 mm allowed 196 binding elements to be located on a single
22 mm square chip, and in turn the chip can easily fit in one well of a
six-well culture plate. The ability of microarrays to localize multiple
experiments to same a solution environment is invaluable for applications
requiring frequent maintenance. For example, cultures of undifferentiated
ES cell require daily changes of growth media. These changes can be
easily performed on the entire microarray in one large well of a cell
culture plate. For comparison, daily maintenance of experiments, on the
same scale, performed in two 96-well plates, while practical, would
require either significant human labor or specialized robotic equipment
to function in a sterile environment to perform daily maintenance.

[0048] The images displayed in FIG. 1 illustrate the results from four
independent experiments utilizing two different hES cell lines (H1 and
H9; WiCell; Madison, Wis.) grown on defined surfaces. Cells grown on
Matrigel® were detached using collagenase treatment and were plated
on the microarray chip in the presence of CM. After twenty-four hours,
media was replaced to remove dead and non-adherent cells. Attached cells
were allowed to proliferate on the chip for five to six days and were
supplement with fresh conditioned growth media daily. The experiment was
stopped once cells reached 100% density in several (but not all) array
elements. If continued, the cells in the array elements presenting 100%
cell density usually detached, yielding square-shaped sheets of cells
floating in solution, while cells in the other elements continued
proliferation (not shown). This observation is consistent with those made
earlier indicating cell growth variability in multi-component arrays and
importance of the balanced duration of the experiment. Once the
experiment was stopped, the cells were fixed and stained for markers of
hES cell pluripotency. Specifically, we tested hES cell line H1 grown in
0.5×0.5 mm elements for the presence of endogenous alkaline
phosphatase (FIG. 1b) and hES cell line H1 grown in 0.8×0.8 mm
elements for the presence of Oct4 transcription factor (FIG. 1c). We have
observed three general types of responses in these screens. Array
elements presenting certain peptides promoted no or very little cell
density at any plating conditions. These peptide regions most likely have
no binding partners on ES cell surfaces. Alternatively, these peptides
might be active for short term adhesion but incapable to initiate the
adhesion-dependent signaling pathways controlling long-term ES cell
proliferation. Several other peptide sequences were identified to yield
reproducible square-shaped colonies, regardless of plating variability of
the cells used for the experiment. These robust growth substrates present
the greatest interest because they are relatively insensitive to
variability in cell density, cell clump size, cell survival and other
stochastic features outlined previously. These peptides are identified in
the results presented in FIG. 1. Finally, the screen identified multiple
peptide sequences exhibiting poorly reproducible ES cell adhesion, which
varied not only from experiment to experiment but even within the same
array. These observations indicate that for some peptides there is a
degree of variability in ES cell survival and growth that can be
different for different growth substrates. Of course, substrates
exhibiting high variability in cell growth can hardly be useful for
robust ES cell growth. On the other hand, identification of these ligands
and their binding partners may be helpful in understanding the molecular
basis for the tremendous heterogeneity in ES cell populations. Overall,
these experiments demonstrated that multi-component arrays can be used
for the identification of surfaces supporting ES cell adhesion and
proliferation. We also show that screens in different growth conditions
can be easily repeated with identical arrays providing valuable
information about robustness of each adhesive surface.

[0049] As discussed above, the majority of other array fabrication
techniques have no control over surface presentation of the surface-bound
peptides, and in many cases, uncontrolled immobilization conditions in
array fabrication can result in decreased activity of the immobilized
ligands. In contrast, the orientatior of the peptides in these SAMs is
highly uniform, and it is dictated solely by structure of the AT used for
the fabrication of the SAM. Moreover, we have utilized facile solid-phase
methodologies to synthesize peptides displaying AT-moiety on their
N-terminus or C-terminus (FIG. 1a), selectively. Spotting of these ATs on
gold yields SAMs displaying the same amino acid sequence in the opposite
orientation (FIG. 1d). Thus, this method provides a means to identify a
mode for presenting a binding peptide that provides optimal interactions
with cells. Data from such experiments also can be used to suggest which
part of the large peptide participates in the interaction with the
receptor. An example of the latter is exemplified by the binding profile
of the SDPGYIGSR (SEQ ID NO:2) sequence. This peptide contains a YIGSR
recognition motif that binds 32/64 kDa laminin receptor expressed at a
low level in ES cells. In our experiments, N-to-C orientation exposing
YIGSR moiety supports cell adhesion for ES cells, albeit with moderate
reproducibility. Switching the surface orientation hinders the YIGSR
region and exposes the SDPG-portion and obliterates cell adhesion in the
same plating environment. Thus, orientation as well as sequence is
important to the cell adhesion and both can be selectively controlled
using the SAM technique described here.

[0050] To test whether our screen can provide immediate information about
the specificity of interactions, we explored the effect of sequence
specificity, overall charge and surface density of a peptide binding site
on its ability to support ES cell binding. The overall charge of the
peptide was controlled by synthesizing the AT-peptides with either free
or acetylated N-terminus. We assumed that the alteration of the charge on
N-terminus in C-to-N orientation would have the most profound effect if
the interaction of peptide with cell is non-specific. The surface density
of the peptides in the SAMs can be easily controlled by co-assembly of
peptide-AT with AT presenting no binding epitope (glucamine-AT, FIG. 1a).
H1 cells were grown on the array presenting gradients of three peptides
(FIG. 1e). We observed that the overall charge has no effect on cell
binding to peptide, and both peptides exhibited cell binding in a similar
range of surface concentrations. In contrast, the specific peptide
sequence presented was critical: A peptide with a scrambled sequence
exhibited negligible cell adhesion at 100% density, and no adhesions at
any surface densities below 100%. These results indicate that the
GRNIAEIIKDI (SEQ ID NO:3) peptide exhibits specific binding. Thus, the
array of SAMs provides a simple means for assessing cell binding
specificity, qualitative affinities and preferred binding orientation for
each screened peptide on the single microchip. Most importantly, this
requires no prior knowledge about the ligand binding mode or its partner
receptor on the ES cell.

[0051] Examination of the cell population within an array element provides
a semi-quantitative indication of differentiation state of the ES cells.
More rigorous analysis involves fluorescence-activated cell sorting
(FACS) measurements to identify what population of the cells express
specific markers of pluripotent stem cells. Therefore, we proliferated
hES cells on gold-coated substrates of larger area (22×22 mm)
containing one-component SAMs. FIG. 2a illustrate a representative FACS
analysis of an ES cell population grown on a synthetic substrate after 1
week. Table 1 outlines the percentages of Oct4 positive cells obtained
from each particular substrate. Not only can cells be grown on these
substrates in the presence of MEF-CM, but some substrates retain their
ability to support ES cell growth in defined growth media. Specifically,
we tested hES cell line H9 grown in synthetic media supplemented with
high concentrations of basic FGF (referred to as UM100, indicating
presence of 100 mg/ml of FGF-2 among the other components). Importantly,
these conditions support growth of undifferentiated hES cells on
Matrigel®. FIG. 2b shows a FACS analysis of ES cells grown on SAMs
presenting DITYVRLKF (SEQ ID NO:4) sequence in UM100 media. The indicated
high level of Oct4 expression confirms their undifferentiated state after
1 week of culture.

[0052] These results demonstrate that SAMs present a rich and flexible
foundation for the fabrication of microarrays. Moreover, SAMs can be
fabricated on large enough areas to grow hES cells in any quantities.
However, their fabrication requires gold-coated surfaces, which makes
them inconvenient candidates for the ultimate growth substrate.
Therefore, we sought to demonstrate that the results of our screen can
also be readily utilized to fabricate a material more flexible in
handling than SAMs on gold, yet exhibiting growth support for
undifferentiated ES cells. This result would also suggest that screens
utilizing a SAM-based array strategy for a particular cellular outcome
(i.e. adhesion, growth, differentiation) can be immediately used as a
starting point for the fabrication of advanced biomaterials exhibiting
similar function.

[0053] To allow a smooth transition from SAM-based screen to a material
design, we aimed for the materials fabrication strategy yielding high
control over peptide ligand presentation. Therefore, we utilized the
approach pioneered by Stupp and co-workers (Hartgerink et al., Proc.
Natl. Acad. Sci. U.S.A. 99, 5133-5138(2002)) to fabricate hydrogels
composed of nanofibers presenting peptide ligands. Like SAMs, cylindrical
nanofibers present peptide ligands or epitopes at a very high density and
with a single, defined orientation. Additionally, we have envisioned that
the control over the peptide orientation in the hydrogel can be readily
achieved through simple solid-phase synthesis. Therefore all aspects of
presentation of peptides in SAMs (FIGS. 1d-e) can be readily translated
to those in hydrogels. To test this hypothesis, we synthesized
amphiphiles presenting the GRNIAEIIKDI (SEQ ID NO:3) sequence in C-to-N
and N-to-C orientation (FIG. 3a). Solutions of either of these peptides
form a gel upon acidification. The formation of nanofiber morphology was
confirmed by TEM. Indeed, this fabricated hydrogel can support adhesion
and proliferation of ES cells, yielding undifferentiated ES population
after one week as confirmed by FACS analysis for pluripotency marker Oct4
(FIG. 3b).

[0055] hES cells (H1B) were passaged using the protease dispase from
Matrigel® to the peptide surface arrays described above containing
either BMP/RGD, BMP/VHB, FGF, FGF/RGD or FGF/VHB at various
concentrations. hES cells were cultured on the arrays for thirteen days,
fixed and stained by immunofluorescence. The cells were stained for a
marker of pluripotency, Oct-4 (Green), and counterstained with DAPI
(Blue). A combination of the FGF peptide mimic and the vitronectin
heparin-binding motif supported hES cell self-renewal--the array element
contained cells expressing Oct-4. Cells on the other array elements no
longer express Oct-4 and are thus no longer pluripotent.

[0056] Synergy Experiment: hES cells (H1B) were passaged using
non-enzymatic methods from Matrigel® to the peptide surface arrays as
described above (containing either RGD, FGF mimic or a combination of RGD
and FGF mimic sequences). hES cells were cultured on the arrays for
twenty-four hours and then fixed and stained by immunofluorescence. The
cells were stained for a marker of pluripotency, Oct-4 (Red), and
counterstained with DAPI (Blue). As opposed to when the peptides are
arrayed individually, a combination of the two peptides promoted stronger
short-term adhesion.

[0057] It is also envisioned that the ligand moieties for the SAMs of the
present invention can include ligands that are other than peptides. We
have tested whether arrays of SAMs can be used to present small organic
molecules different from peptides and whether these arrays can be used
for functional cell-based screening. We have synthesized three alkane
thiols presenting ligands for avb3 integrtins. For integrin ligands, we
used a peptide (linear RGD containing peptide), a cyclic inhibitor
molecule containing elements of RGD peptide sequence and a known
non-peptide integrin inhibitor (FIG. 7). The latter two molecules were
synthesized by a straightforward synthetic strategy Eased on coupling of
inhibitor molecules presenting amine-linker and protected alkane thiol
moiety containing amine linker using squaric acid linkage (FIG. 8). Thus,
while peptides may be used to test ligands, the ultimate ligands which
might be used to host cells can be non-peptide ligands that provide
similar signals to the cells on the array.

[0058] Methods and Materials for SAM Synthesis and Assembly

[0059] Materials: All reagents for solution phase chemical synthesis were
purchased from Aldrich (Milwaukee, Wis.) and used without further
purification with the following exception: 2,2'-azobisisobutyronitile
(AIBN) was recrystallized from acetone prior to use. Amino acids were
protected with 9-fluorenylmethyloxycarbonyl (Fmoc) and
4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy (Rink amide AM) resin
was used for the solid phase peptide synthesis (SPPS) and were purchased
from NovaBiochem (San Diego, Calif.). N-Hydroxybenzotriazole (HOBt) was
purchased from Advanced ChemTech (Louisville, Ky.). Anhydrous
tetrahydrofurane (THF) was distilled from Na-benzophenone ketyl.
N,N-dimethylformamide (DMF) was vacuum distilled from 4 Å molecular
sieves. UV-irradiation at 254 nm was done in a Rayonet Photochemical
Chamber Reactor, Model RPR-200. Flash chromatography was performed with
silica gel 60, 230-450 mesh (Sorbent Technologies). Preparative HPLC was
performed on a Spectra system P2000 instrument with a UV2000 detector.
Preparative HPLC conditions used: Vydac® 150 cm×22 mm C-18
reverse-phase column flowing at 10 mL/min using 0.1% TFA as mobile phase
A and 0.1% TFA in acetonitrile as mobile phase B. Electrospray ionization
(ESI) high resolution mass spectra (HRMS) were obtained with a Micromass
LCT®. H-NMR and C-NMR were recorded on a Bruker AC-300. The liquid
chromatrograph/mass spectrograph (LCMS) used was a Shimadzu LCMS-2010
instrument with photodiode array detector (SPD-M10Avp), and a single
quadrupole analyzer. HPLC conditions used for LCMS were: Supelco
(Bellefonte, Pa.) 15 cm×2.1 mm C-18 wide-pore reverse-phase column
flowing at 200 μL/min using 0.4% formic acid as mobile phase A and
0.2% formic acid in acetonitrile as mobile phase B. All tissue culture
reagents were obtained from Gibco/Invitrogen (Carlsbad, Calif.). The cell
lines SH-SY5Y and Swiss-3T3 were obtained from American Type Culture
Collection (ATCC, Manassas, Va.) and were passage 20 or lower. Vector Red
Alkaline Phosphatase Substrate Kit I was obtained from Vector Labs
(Burlingame, Calif.). Glass coverslips (Corning No 11/2, 23 mm squares)
for the array production were purchased from Fisher.

[0060] Scheme 1: The molecules are illustrated in FIG. 5, and this scheme
involves the synthesis of the AT peptide conjugates. The general strategy
employed was adapted from Houseman et al. with some modifications in the
synthetic steps. In addition the tetra(ethylene glycol) derivative was
employed rather than hexa(ethylene glycol). Abbreviations used: TFA,
trifluoroacetic acid; TIS, triisopropylsilane; EDT, ethanedithiol; PhOH,
phenol.

[0066] Peptide-ATs. Peptides were synthesized on a Pioneer® Peptide
Synthesis System using standard Fmoc chemistry on Rink Amide AM resin
(loading: 0.56 mmol/g). Peptide-alkanethiol conjugates were prepared
similarly to the procedure of Houseman et al. Briefly, resin containing
protected peptide with a free N-terminus was swollen in dry THF, 5-fold
excess of each of the compound 1, HOBt and 1,3-diisopropylcarbodiimide
(DIC) was added to the resin suspension in THF. The resin was incubated
for 12 hours and another 3-fold excess of DIC and HOBt was added. After 3
hours, the resin was tested with Kaiser test, washed with DMF and
dichloromethane and dried in vacuo. After cleavage with
TFA/DIC/EDT/H2O/phenol (36:1:1:1:1) for 2 hours and ether
precipitation, conjugates were purified by preparative HPLC. Gradient
used (percentage of mobile phase A): 100→0% 20 min, 0% 3 min.
0→100% 3 minutes. Peaks at retention time around 17 minutes were
collected. Each purified sample was analyzed by LCMS and H NMR. Note: The
presence of triplet of 1,1,1-triples δ 2.49 (t[111t], 2H, J=7.1 Hz,
JHD=1.0 Hz) in H NMR at AT-peptides in CD3OD is indicative of
free thiol functionality. It is the signal of methylene hydrogens next to
the free thiol functionality (7 NZ, coupling to neighboring methylene, 1
Hz coupling to deuterium on free thiol).

[0075] (12,12,13,13,14,14,15,15,16,16,17,17,18,18,19,19-Heptadecafluoro-no-
nadecane-1-thiol)(Fluoro-AT): This synthesis was performed according to
the procedure of Graupe et al. with the exception that LiAlH4 was
used to reduce the alkyl-iodide and thioester moiety instead of the
published NaBH4.